Project Summary This MIRA proposal focuses on two overlapping areas: stress response regulation and the functions of redox-based signaling in vivo. It is a fundamentally important problem how organisms detect and respond to different forms of stress. Much has been learned in this area but we still have a very incomplete understanding of how some stresses are detected, including reactive small molecules such as ROS. For many years my group has studied stress responses and aging in C. elegans, focusing on the Nrf2 transcription factor ortholog SKN-1. Nrf2 mediates a conserved detoxification response to reactive small molecules but has many additional functions, and is of great importance in health and disease. Working in C. elegans we have defined a number of aspects of SKN-1/Nrf2 regulation and functions, including its major role in longevity assurance. We have recently uncovered an exciting mechanism of SKN-1/Nrf regulation that forms the basis for this new research direction. We find that SKN-1 and human Nrf2 are activated at the ER by a localized ROS signal that can derive from the ER, NOX enzyme activation induced by stress, or mitochondria. This signal induces sulfenylation of a single Cys within the kinase activation loop of the ER unfolded protein sensor IRE-1, resulting in acute inhibition of the IRE-1 unfolded protein response and activation of p38 signaling at IRE-1 through a second sulfenylation event. p38 signaling in turn activates SKN-1/Nrf2. Remarkably, other kinases of major interest (AKT, p70S6K, ROCK1) seem to be regulated through sulfenylation of the same Cys. The data reveal an unexpected IRE-1 function that is regulated by a redox switch, a major stress sensor for SKN- 1/Nrf2, and a possible rationale for how redox stress can affect so many cellular processes. They also suggest that the scope and functional versatility of Cys-based signaling are much wider than is generally appreciated. In our proposed research we will continue to identify mechanisms of SKN-1/Nrf2 regulation and their functions in vivo, but will also cast our net wider in utilizing the advantages of C. elegans to explore mechanisms and functions of Cys redox signaling in the context of stress, growth, and other conditions. We will refine models for IRE-1 regulation of SKN-1/Nrf2 and its functions in vivo. We will also similarly study SKN- 1/Nrf2 regulation by the chaperone TRIC, another mechanism we have identified that may involve redox signaling, and build upon screening results to develop new models for SKN-1/Nrf2 regulation. Using mass spectrometry (MS), we will collaboratively identify C. elegans proteins that are Cys-sulfenylated under stress and growth conditions. We will investigate regulatory and in vivo implications of this modification for the kinases indicated above, and candidates chosen from our MS data. C. elegans will be ideal for this work because of the relative rapidity of Cas9/CRISPR genome editing, and phenotypic analyses. Our research will reveal stress-responsive regulatory mechanisms of fundamental interest, and take major steps towards identifying new mechanistic targets and functional implications of redox signaling in vivo.